Abstract
Suspensions of hemoglobin microparticles (HbMPs) are promising tools as oxygen therapeutics. For the approval of clinical studies extensive characterization of these HbMPs with a size of about 750 nm is required regarding physical properties, function, pharmaco-kinetics and toxicology. The standard absorbance measurements in blood gas analyzers require dissolution of red blood cells which does not work for HbMP. Therefore, we have developed a robust and rapid optical method for the quality and functionality control of HbMPs. It allows simultaneous determination of the portion of the two states of hemoglobin oxygenated hemoglobin (oxyHb) and deoxygenated hemoglobin (deoxyHb) as well as the content of methemoglobin (metHb). Based on the measurement of collimated transmission spectra between 300 nm and 800 nm, the average extinction cross section of HbMPs is derived. A numerical method is applied to determine the composition of the HbMPs based on their wavelength-dependent refractive index (RI), which is a superposition of the three different states of Hb. Thus, light-scattering properties, including extinction cross sections can be simulated for different compositions and sizes. By comparison to measured spectra, the relative concentrations of oxyHb, deoxyHb, metHb are accessible. For validation of the optically determined composition of the HbMPs, we used X-ray fluorescence spectrometry for the ratio of Fe(II) (oxyHb/deoxyHb) and Fe(III) (metHb). High accuracy density measurements served to access heme-free proteins, size was determined by dynamic light scattering and analytical centrifugation and the shape of the HbMPs was visualized by electron and atomic force microscopy.
Highlights
Worldwide, about 85 million units of red blood cells are transfused annually [1] and an increasing need for blood is observed
Hemoglobin and albumin molecules were cross-linked by glutaraldehyde with a final concentration of 0.04%. and the salt templates cross-linked by glutaraldehyde with a final concentration of 0.04%. and the salt templates were dissolved by 0.18 mol L−−11 ethylenediaminetetraacetic acid (EDTA) resulting in the final hemoglobin microparticles (HbMPs)
Results for the metHb fraction in HbMPs obtained by spectral extinction measurements and Near edge X-ray absorption fine structure (NEXAFS) are compared to measurements based on oxygen release [23,24,25], details of which are given in Materials and Methods section
Summary
About 85 million units of red blood cells are transfused annually [1] and an increasing need for blood is observed. The transfusion of red blood cells holds risks and limitations. Mismatched transfusions as well as transmissible infections present a major health hazard [2]. The storage time of red blood cells is limited, cold chain principles must be respected and the logistics are highly demanding. There is a highly and fast growing demand for an artificial oxygen carrier which is able to carry and deliver oxygen at the sites of need. The search for an artificial blood substitute based on oxygen carriers has presented a great challenge in transfusion medicine for more than three decades. In the focus of research are hemoglobin-based oxygen carriers (HBOCs) [3].
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